! Aggregates of alpha-synuclein (?Syn) are a pathological hallmark of Parkinson's disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). Moreover, genetic increases in ?Syn expression and point mutations in the ?Syn gene are an increasingly well-documented precipitant of early- onset familial PD (fPD). Pathological oligomers have been extensively studied, but only detailed analyses of synthetic aggregates from recombinant sources are available. Therefore, we wish to characterize pathological ?Syn species we are isolating from PD/DLB and MSA brains. We plan here an extensive characterization of toxic and self-permissive aggregated forms of ?Syn, isolated from human patient brain. Characterization of the isolated species will be both functional (toxicity) and structural for a correlative analysis of structure-function relationship. Additional analysis of different brains will allow a comparison between structure specificity of the different diseases (disease ?strains?). The isolation procedure will focus on gentle, non-denaturing methods targeting 3 different sub-cellular locales (cytosol, membrane- associated, insoluble). Results from our novel concept could point to exciting possibilities in rational drug design based on exact structural details of pathogenic strains. Detection and quantification the pathogenic ?Syn oligomers may be used as a biomarker for diagnosis of synucleinopathies and will provide novel reagents to the community along with valuable platforms (strain specific PMCA assays) for therapeutic compound screening. To move our hypotheses forward, we propose to gather data in two major directions: Aim 1: Search for the existence of natively soluble `pathological seeds' of ?Syn in human brain homogenates of PD, DLB and MSA patients under non-denaturing conditions and quantify their bioactivity in dynamic assays of pathogenic aggregation and toxicity. Aim 2: Characterize the unique structural features (?strains?) of the brain isolated insoluble aggregates from each disease subgroup (PD/DLB/MSA) and their amplification products to contrast their biochemical and biophysical properties in relation to disease. Results from our novel concept could point to exciting possibilities in rational PD drug design based on exact structural details of pathogenic strains. In addition, detection and quantification the pathogenic ?Syn oligomers may be used as a biomarker for diagnosis of synucleinopathies and will provide novel reagents (synthetic human brain derived strains) to the community along with valuable platforms (strain specific detection assays) for therapeutic compound screening. ! !